Session H35: Emerging Spectroscopic Techniques

Real-time detection of multiple biomolecular reactions on a functionalized glass surface using a scanning oblique-incidence optical reflectivity difference (an ellipsometric technique).

One of the enabling platforms in proteomic research is parallel (high-throughput) detection of multiple biomolecular interactions on a microarray. To keep conformational and in turn functional integrity of protein molecules, label-free detection is desirable. We have developed an oblique-incidence optical reflectivity difference (OI-RD) technique for label-free measurements of protein reactions with molecular targets in microarray format immobilized on functionalized glass surface. As an ellipsometric technique, OI-RD measures changes in thickness and/or optical dielectric response instead of fluorescence. By incorporating total internal refection geometry and a multi-element photodiode array detector, we demonstrate how such the OI-RD technique can be efficiently used to measure multiple protein reactions in real time with surface-immobilized molecules or molecular groups on a glass substrate.   

In Situ X-ray Reflectivity Studies of Protein Adsorption onto Functionalized Surfaces

The adsorption of protein films onto solid surfaces, both artificial and naturally occurring, have been widely studied using a variety of techniques due to their importance in medicine, biomedical applications, and the general understanding of protein structure and function. What have yet to be performed are in situ, time-resolved, high-resolution structural studies of these systems. We have begun a project that uses the technique of in situ x-ray reflectivity to obtain highly resolved structural information with time resolution on the order of minutes. This talk will present our first findings of serum albumin and immunoglobulin G films on hydrophobic self-assembled monolayers. The protein films are readily observable, showing extensive denaturing after adsorption with a slow decay of density into the aqueous solution. Additionally, a thin low-density region that occurs between the hydrophobic film and the solution persists after protein deposition. Comparisons to films that are removed from solution, the influence of solution concentration, the effects of x-ray damage, and the time scales for protein film formation and evolution will also be discussed.   

Mid-IR spectra of the bio-related molecules in the gas phase

Mid-IR spectra of gas phase bio-related molecules R-OH, R-COOH and simple non-aromatic amino acids, such as glycine and valine, detected by vacuum ultraviolet (VUV), 10.5 eV single photon ionization of supersonically expanded and cooled samples, are presented and discussed. Molecules and their fragment species, generated by a proton transfer reaction following ionization, are identified by time of flight mass spectroscopy. The fundamentals and overtones of the CH and OH stretches and some combination bands are identified in the spectra. Rotational resolution for the OH mode and its first overtone yield an estimate of $\sim $50 K for the methanol monomer in the supersonic beam. Two neutral C$_{2}$H$_{5}$OH conformers can be identified by high sensitivity IR plus VUV nonresonant ionization and fragmentation detected (NRIFD-IR) vibrational spectroscopy. Free OH and NH stretches are missing in the spectrum of glycine and valine, indicating that the strong intra-molecular hydrogen bonds are formed in these gas phase species.   

Single Quantum Dots Imaged with Resonance Rayleigh Scattering Do Not Blink

Semiconductor quantum dots have become a robust fluorescent marker for the life sciences. Two key issues limit the broad use of quantum dots as fluorescent markers: heterogeneous emission and non-radiant dark populations. All bright quantum dots blink stochastically, have considerable heterogeneity in their emission, and have fluctuations in their fluorescence lifetimes, limiting their utility as single particle trackers by introducing potentially large interruptions in particle trajectories. Further, a significant fraction does not fluoresce at all, undermining biophysical studies such as immuno-fluorescence. We present an alternative or complement to fluorescent imaging of quantum dots. We have developed a new technique, resonant Rayleigh scattering (RRS) microscopy, for imaging single quantum dots which does not exhibit blinking. Detection of individual quantum dots, both surface immobilized and freely diffusing in aqueous solution, is demonstrated. Non-fluorescent populations of quantum dots are visible through RRS microscopy. Though other non-fluorescence detection techniques exist they are significantly more complicated than our technique, which requires minimal alteration of a conventional confocal fluorescence microscope.   

Development of 0.24 THz pulsed electron paramagnetic resonance to ``film'' proteins in action with the UCSB free electron laser

Pulsed electron paramagnetic resonance (EPR) is extremely useful to study the fast dynamics of molecules. Currently, most high-power pulsed EPR experiments are performed near 10 GHz, with a time resolution of 100 ns. The spin dephasing times of spin labels on proteins in aqueous solution are tens of ns. Thus, conventional pulsed EPR measurements of proteins are performed on frozen samples. There exist instruments which operate at 95 GHz with time resolution shorter than 100 ns. We present the development of a 0.24 THz pulsed EPR system which is expected to have sub-ns time resolution, enabling the EPR study of proteins in solution. The system uses the UCSB free electron laser (FEL) to produce kW-level pulses at 240 GHz. A ``pulse-slicer'' shortens the FEL's microsecond pulses to the ns range. Sequences of two or three pulses separated by up to 25 ns will be made using a home-made delay line. A superheterodyne detection system is being fabricated to be sensitive enough to detect 1nW signals and also protected from kW FEL inputs.   

Three-Dimensional Imaging of Single Large Macromolecules Using Equally Sloped Tomography

We report the development of equally sloped tomography for the reconstruction of the 3D structure of single large macromolecules. In a combination of pseudo-polar fast Fourier transform and the oversampling method with an iterative algorithm, equally sloped tomography makes superior 3D reconstruction to conventional tomography which has an intrinsic drawback due to the use of equally angled 2D projections. By employing equally sloped tomography and cryo electron microscopy, we have obtained the 3D structure of single hemocyanin protein molecules and HIV viruses at $\sim$ 5 nanometer resolution. Preliminary analysis based on cross- correlation has indicated that the 3D images using equally sloped tomography are superior to those of the conventional method. We believe this general approach will find broad applications in high-resolution 3D imaging of large macromolecules.